Living cells must continuously sense and respond to a broad spectrum of intra- and extra-cellular inputs. This information must be processed so as to produce a response that is appropriate, comprehensive, and efficient. This complex task requires the intimate integration of signal transduction 'cascades' into computationally-sophisticated networks. Protein phosphorylation-dephosphorylation processes constitute prominent, core components of such networks. In mammalian cells the task of understanding regulatory networks as complete systems is confounded by the multiplicity (103-104) and redundancy of their components. A clear need exists for vehicles to permit study of the protein phosphorylation-dephosphorylation networks as integrated systems on a smaller scale in the near term. Such vehicles will serve as pathfinders for the analysis of more quantitatively complex organisms and will trace the history of their development. The objective of this proposal is to map the protein O-phosphorylation [i.e. those events targeting the hydroxyl side chains of serine,threonine, and/or tyrosine] network of the cyanobacterium Synechocystis sp. PCC 6803, a biochemically complex and environmentally adaptable organism. This cyanobacterium contains a quantitatively tractable protein O-phosphorylation network (approximately 102) that prominently features homologs of 'eukaryotic' protein kinases, protein- serine/threonine phosphatases, and protein-tyrosine phosphatases. Synechocystis sp. PCC 6803 is genetically malleable and its complete genome sequence is known. The specific aims of the study outlined herein are: 1. To identify the proteins in Synechocystis sp. PCC 6803 that undergo modification via phosphorylation of serine, threonine, and/or tyrosine residues. 2. To identify the serine, threonine, and tyrosine-specific protein kinases and protein phosphatases in this organism. 3. To identify physiologically-relevant enzyme-substrate relationships between the phosphoproteins indentified in aim 1 and the protein kinases and protein phosphatases identified in aim 2. The realization of these aims will contribute to our long-term goal of mapping and modeling the molecular interplay of a complete signal transduction/regulatory network on a cellular scale.